1. Field of the Invention
This invention relates to diagnostic assays useful for identifying, classifying and monitoring cancer patients for Bcl-2 family inhibitor therapy, and in particular relates to measurement of certain markers that can identify patients whose cancer is likely to be resistant to most Bcl-2 inhibitors.
2. Description of Related Art
Genetic heterogeneity of cancer is a factor complicating the development of efficacious cancer drugs. Cancers that are considered to be a single disease entity according to classical histopathological classification often reveal multiple genomic subtypes when subjected to molecular profiling. In some cases, molecular classification proved to be more accurate than the classical pathology. The efficacy of targeted cancer drugs may correlate with the presence of a genomic feature, such as a gene amplification (Cobleigh et al., 1999; Lynch et al., 2004). For Her-2 in breast cancer, it has been demonstrated that detection of gene amplification provides superior prognostic and treatment selection information as compared with the detection by immunohistochemistry (IHC) of the protein over-expression (Pauletti et al., 2000). A need therefore exists for genomic classification markers that may improve the response rate of patients to targeted cancer therapy.
Lung cancer is an area of active research for new targeted cancer therapies. Lung malignancies are the leading cause of cancer mortality, approximately 160,000 deaths in the United States in 2006. Small-cell lung carcinoma (SCLC) is a histopathological subtype of lung cancer, which represents approximately 20% of lung cancer cases. The survival rate for this subtype is low (long-term survival 4-5%) and has not improved significantly in the past decade, despite the introduction of new chemotherapy regimens. The remainder of lung cancer cases are non-small-cell lung carcinomas (NSCLC), a category which is comprised of several common subtypes. In the past several years, there has been substantial progress in the development of targeted therapies for NSCLC, such as erlotinib and gefitinib. Genomic biomarkers have been discovered which enable stratification of NSCLC patients into potential responders and non-responders. In particular, mutations and amplifications in the EGFR kinase domain were shown to correlate with the response to erlotinib and gefitinib. Unfortunately, no such progress has been achieved with SCLC, even though genomic analysis of SCLC cell lines and tumors has been reported (Ashman et al., 2002; Coe et al., 2006; Kim et al., 2006).
Targeted cancer therapy research has been reported against members of the Bcl-2 protein family, which are central regulators of programmed cell death. The Bcl-2 family members that inhibit apoptosis are over-expressed in cancers and contribute to tumorigenesis. Bcl-2 expression has been strongly correlated with resistance to cancer therapy and decreased survival. For example, the emergence of androgen independence in prostate cancer is characterized by a high incidence of Bcl-2 expression (>40% of the cohort examined) (Chaudhary et al., 1999), which also corresponds to an increased resistance to therapy. Furthermore, over-expression of Bcl-2 in both NSCLC and SCLC cell lines, has been demonstrated to induce resistance to cytotoxic agents (Ohmori et al., 1993; Yasui et al., 2004). Yasui et al. (2004) describe copy number gain at the Bcl-w (BCL2L2) locus and conclude that Bcl-w expression is at least partially responsible for the chemoresistance of SKOV3/VP. Yatsui et al. (2004) does not disclose identification of Bcl-2 family copy number change in any other cancer cell line.
Martinez-Climent et al. (2003) describe identification of a copy number change at 18q21, including the Bcl-2 locus, in the transformation of follicular lymphoma to large cell lymphoma (Martinez-Climent et al., 2003). Monni et al. (1996) report multiple copy number changes in diffuse large B-cell lymphoma (Monni et al., 1996). Galteland et al. (2005) report gain of the chromosome locus of Bcl-2 in B-cell non-Hodgkin's lymphomas (Galteland et al., 2005). Nupponen, et al (1998) describe low level copy number gain of Bcl-2 in four of 17 samples of recurrent prostate cancer (Nupponen et al., 1998). Olejniczak et al. have reported a gain on 18q21 in SCLC that contained the Bcl-2 gene and was associated with sensitivity to a Bcl-2 inhibitor ABT-737 (Olejniczak et al., 2007).
A key area of oncology research today is the study of acquired resistance to targeted therapies. Targeted therapies provide undeniable benefits to patients; however, in most cases, these benefits are temporary, as tumors exact numerous strategies to escape the effects of these therapies. These resistance mechanisms are complex and diverse. Once understanding these mechanisms is achieved, new therapies that target resistance mechanisms can be developed and deployed.
BH3 mimetics are highly targeted compounds engineered to trigger apoptosis by inhibiting specific anti-apoptotic Bcl-2 family members. Resistance to the effects of these drugs may arise during treatment.
One such mimetic, ABT-737 (oral form, ABT-263), is a small-molecule inhibitor of the Bcl-2 family members Bcl-2, Bcl-xL, and Bcl-w, and has been shown to induce regression of solid tumors (Oltersdorf et al., 2005). ABT-737 has been tested against a diverse panel of human cancer cell lines and has displayed selective potency against SCLC and lymphoma cell lines (Oltersdorf et al., 2005). ABT-737's chemical structure is provided by Oltersdorf et al. (2005) at p. 679.
In many tumors, the capacity of the Bcl-2 family to remove damaged cells is subverted, either because a pro-survival family member is over-expressed (Cory et al., 2003), or because mutations in the p53 pathway halt p53-mediated induction of the BH3-only proteins that would otherwise trigger apoptosis (Jeffers et al., 2003; Shibue et al., 2003; Villunger et al., 2003). Interestingly, however, even when resistant to Bcl-2 inhibitor family's effects, the apoptotic machinery in tumor cells remains intact.
In one study of cells that over-expressed pro-survival Bcl-2 family members, the cells were found to be resistant to ABT-737, which usually triggers Bax/Bak-mediated apoptosis. Van Delft et al. (2006) found that the cells were chemo-refractive to ABT-737 because another pro-survival relative, Mcl-1, was over-expressed. By down-regulating Mcl-1, the cells became sensitive to ABT-737-mediated apoptosis (van Delft et al., 2006). The role of Mcl-1 in rendering a cancer cell refractory to ABT-737's effects was demonstrated by Tahir et al. (2007) (Olejniczak et al., 2007). In that study, Tahir et al. (2007) observed that as a small-cell lung cancer line was exposed to escalating doses of ABT-737, the cells acquired ABT-737 resistance, which was associated with up-regulation of Mcl-1 expression.
Thus there is a need to be able to identify cells that are resistant to antagonists of Bcl-2 family members.